Although the gammadelta T cells have been shown to have immunoregulatory effects in a number of experimental systems, their role in immunobiology is still not clear. Surprisingly, it is possible to divide gammadelta T cells into functional subsets based on the Vgamma and/or Vdelta elements they express. Based on such findings, we have hypothesized that certain inducible host molecules act as ligands for gammadelta T cell receptors (TCRs), and are important in eliciting their immunoregulatory effects. However, the nature of the ligands recognized by gammadelta TCRs remains controversial. Furthermore, recent findings have suggested that gammadelta T cell responses can be evoked by molecules other than the TCR, such as chemokine receptors or the Toll-like receptors of the innate immune system, making experiments in which cellular activation is used as a readout for TCR-mediated stimulation difficult to interpret. We believe that the gammadelta T cell receptor itself may hold the key to elucidating the role of the TCR in bringing about gammadelta T cell function. In this proposal, we present preliminary evidence that multimerized versions of mouse soluble gammadelta TCRs can be used like monoclonal antibodies to stain cells bearing their putative ligands. The specific aims of this proposal make use of such soluble TCRs to further investigate the natural ligands of gammadelta TCRs: Aim 1 - to investigate properties of natural ligands for gammadelta TCRs. Using labeled versions of four different gammadelta soluble TCRs (sTCRs), we will use flow cytometry in an attempt to detect the ligands in thymus and certain peripheral organs that drive the development or differentiation of distinct gammadelta T cell subsets. Second, using sTCRs from a subset whose TCRs are variable, we will determine whether different members of a given subset can recognize the same ligand. Third, we will attempt to determine if these ligands are MHC class I or class I-like by examining cells from particular mutant mouse strains. Aim 2 - to purify and identify natural ligands for gammadelta TCRs. Using two different approaches, we will attempt to isolate and identify the natural ligand for the V?6/Vd1 invariant TCR, as well as for a typical polymorphic Vgamma1/Vdelta6.3 TCR. The first approach involves screening cDNA expression clones for their ability to stain with either the Vgamma6/Vdelta1 or Vgamma1/Vdelta6.3 sTCR. The backup approach involves generation of ligand specific mAbs, identified by their ability to block sTCR/ligand binding, as reagents to affinity-purify the ligand.